1. Field of the Invention
This invention relates to the field of detecting nucleotide variations in a nucleic acid. More particularly, the invention relates to methods of detecting nucleotide variations in a nucleic acid by generating variable length copies of a nucleic acid from a sample, hybridizing those generated nucleic acids to reference nucleic acids, and detecting the presence or absence of nucleotide variations at the 3xe2x80x2-terminal position or the penultimate 3xe2x80x2-position on the variable length nucleic acids.
2. Background Art
The number of diseases that are linked to gene mutations continues to increase as the sequence of the human genome is unraveled. Nucleic acid sequencing is the ultimate standard for detecting nucleotide variations. Nucleic acid sequencing is well suited for detecting unknown mutations or polymorphisms that may occur at any base within a target nucleic acid segment. The chemistry of enzymatic DNA sequencing, the most commonly used method, has essentially remained the same since its conception (Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977)). The art has been improved by technology that has allowed for its automation such as the introduction of fluorescent dyes, robotics and improved electrophoretic systems with automated detection. However, if genetic variations occur at a low frequency in the sample population, automation comes at a cost that is too high for most laboratories. Even in a manual mode, sequencing can be cost prohibitive because it is labor intensive. Thus, there is a need in the art for a simple inexpensive process to screen nucleic acids for unknown nucleotide variations prior to sequencing.
That need in the art is evident by the number of methods being developed to screen for unknown mutations. Single strand conformation polymorphism (SSCP) detects mutations in an unknown sample by comparing its migration rate in a single stranded state to a known sample in a non-denaturing gel, as disclosed by Orita et al., Genomics, 5:874-879 (1989). Changes in nucleotide sequence affect the secondary structure or conformation of a DNA molecule which may alter its migration rate during electrophoresis. This technique, however, is limited to small targets less than 200 bp, has limited sensitivity, and requires rigid electrophoresis conditions to be reproducible. Improvements in SSCP analysis such as dideoxy fingerprinting, both unidirectional (Sarkar et al., Genomics, 13:441-443 (1992)) and bidirectional (Liu et al., Hum. Mol. Genet., 5:107-114 (1996)), and restriction endonuclease fingerprinting (Liu and Sommer, Biotechniques, 18:470-477 (1995)) can detect mutations over a 1 kb span but sacrifice sensitivity for simplicity since the complex pattern of DNA bands generated by these processes makes it difficult to readily detect mutations.
Another method that is used for screening for nucleotide variations in a nucleic acid is based on the differential mobility of heteroduplex molecules as they migrate through a gel matrix. In its simplest form called heteroduplex analysis, an uncharacterized DNA segment, usually an amplification or PCR product, is mixed with the corresponding wild type segment, heated, and allowed to slowly renature, as first described by Nagamine et al. (Am. J. Hum. Genet., 45, 337-339 (1989)). If the uncharacterized nucleic acid has a different sequence than the wild type sequence, heteroduplex molecules are formed. Base mismatches in the heteroduplex alter its migration rate allowing it to be partially resolved from the homoduplex in a non-denaturing gel.
A more sensitive approach called denaturing gradient gel electrophoresis (DGGE) subjects heteroduplex molecules to increasing levels of denaturant in a gradient gel format, as first described by Fisher and Lerman. (Proc. Natl. Acad. Sci. U.S.A., 80:1579-1583 (1983)). As the heteroduplex molecules migrate through the denaturant, they begin to melt, or denature. At this point migration is slowed and is no longer linear. The melting point is slightly different for homoduplex molecules, allowing partial resolution of heteroduplex molecules. Precise control of field strength, temperature and time are critical to achieving reproducible results, and difficult to consistently reproduce.
With constant denaturing gel electrophoresis (CDGE), these variables are less critical since the concentration of denaturant is the same throughout the gel (Hovig et al., Mut. Res., 262:63-71 (1991)). A significant limitation of this technique is that a nucleic acid segment may have more than one melting domain for which separate gels at different denaturant concentrations must be run.
Temporal temperature gradient gel electrophoresis (TTGE) seeks to circumvent this problem by gradually increasing the temperature during electrophoresis, as described by Borresen et al. (Bioradiations, 99:12-113 (1997)). This is a hybrid technique between CDGE and temperature gradient gel electrophoresis which uses temperature only as a denaturant (Rosenbaum and Riesner, Biophys. Chem., 26:235-246 (1987)). As expected, however, this technique is also difficult to perform and also difficult to reproduce.
A recently introduced technique called base excision sequence scanning (BESS) improves upon dideoxy fingerprinting with ddTTP by obviating the need for a separate sequencing reaction (Epicentre Technologies, Madison, Wiss.). The target of interest is amplified by PCR using a labeled primer and a limiting amount of dUTP. After amplification, the products are treated with uracil DNA glycosylase to cleave at uracil sites. Denaturing gel electrophoresis of the fragments then produces a ladder almost identical to a dideoxy T sequencing ladder. The technique is useful for screening DNA segments up to 1 kb for mutations, but is limited by the resolution of gel electrophoresis and it does not detect G to C transversions or vice versa.
Another recently introduced technique uses a structure specific endonuclease called cleavase to digest intrastrand structures and produce fragment length polymorphisms (CFLP) and is described by Brow et al., J. Clin. Microbiol., 34:3129-3137 (1996). The structures are created by denaturing a segment of DNA and then quickly cooling it to the digestion temperature and adding the enzyme. The folding pattern for a given segment may be altered by sequence variations that upon digestion with the enzyme produces a unique banding pattern on a denaturing gel. This technique, however, is severely limited by the resolution of the gel electrophoresis and the complex pattern of DNA bands generated by the process which makes it difficult to detect mutations.
Detection of mutations by chemical or enzymatic cleavage of base pair mismatches in heteroduplex DNA has been described by Noack et al., Proc. Natl. Acad. Sci. U.S.A., 83:586-590 (1986), Cotton et al. Proc. Natl. Acad. Sci. U.S.A., 85:4394-4401 (1988), Cotton et al., U.S. Pat. No. 5,202,231, (Winter et al., Proc. Natl. Acad. Sci. U.S.A., 82:7575-7579 (1989), Myers et al., Science, 230:1245-1246 (1985)), (Lu and Hsu, Genomics, 14:249-255 (1992),) and U.S. Pat. No. 5,698,400. Many of these techniques are limited by the inability of the cleavage reagents to recognize all types of base pair mismatches, and for others this can be overcome by analyzing both strands of a DNA segment. To date, widespread use of these techniques has not been observed, partly because they require highly toxic reagents and the procedures are difficult to perform.
The miniaturization of the DNA hybridization process onto a small solid surface, known as a DNA chip or micro array, allows the analysis of DNA segments without gel electrophoresis. See Macevicz, U.S. Pat. No. 5,002,867, Drmanac., U.S. Pat. No. 5,202,231, Lipshutz et al., Biotechniques, 9(3):442-447 (1995) and Chee et al., Science, 274:610-614 (1996). The resolution of gel electrophoresis, however, strictly limits the size of the DNA segment that can be analyzed for all of the aforementioned mutation detection technologies including DNA sequencing and the high cost of the equipment,and chips used in this process limit its wide spread use.
The present invention provides needed improvements over these prior art methods by providing methods which can detect all possible base variations including single and multiple base substitutions, insertions and deletions. These variations may occur at one or more sites and affect one or more nucleotides at each site for a given locus. Secondly, as a screening process, these methods provide a clear positive or negative result. Thirdly, the process is not limited by the resolution power of gel electrophoresis and therefore allowing the analysis of DNA segments greater that 1 kb in size. Lastly, by way of eliminating electrophoretic detection, it is highly amenable to automation and therefore suitable for high volume screening.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a method of detecting nucleotide variation within a first nucleic acid, comprising generating a set of single-stranded extension products from a first nucleic acid in the presence of modified nucleotide bases, wherein the extension products incorporate modified nucleotides and thereby limit exonuclease activity to the 3xe2x80x2-terminal nucleotide base, and wherein the extension products have variable lengths, hybridizing the variable length extension products to a reference nucleic acid, contacting the hybridizing nucleic acids with an enzyme which can remove and replace the 3xe2x80x2-terminal nucleotide of the extension products in the presence of selected labeled nucleotides, wherein extension products that terminate with a 3xe2x80x2-nucleotide that does not hybridize with the corresponding position on the reference nucleic acid are replaced with a nucleotide that hybridizes with the corresponding nucleotide on the reference nucleic acid and wherein those extension products that had a non-hybridizing nucleotide at the 3xe2x80x2-terminus can now be distinguished from those extension products that had a hybridizing nucleotide at the 3xe2x80x2-terminus, distinguishing those extension products that had a non-hybridizing nucleotide at their 3xe2x80x2-terminus from those extension products that had a hybridizing nucleotide at their 3xe2x80x2-terminus, thereby detecting nucleotide variation in the first nucleic acid.
The invention also provides a method of detecting within a first nucleic acid the presence of a nucleotide variation, comprising generating a set of single-stranded extension products in the presence of modified nucleotide bases, wherein the extension products incorporate modified nucleotides that limit exonuclease activity to the 3xe2x80x2-terminal nucleotide base, and wherein the extension products have variable lengths, hybridizing the variable length extension products to a reference nucleic acid, contacting the hybridizing nucleic acids with an enzyme which can remove and replace the 3xe2x80x2-terminal nucleotide of the extension products in the presence of a selected modified nucleotide which is resistant to further replacement and when incorporated into an extension product inhibits further extension of the extension product, wherein extension products that terminate with a non-modified nucleotide can be further extended and thereby distinguished from those extension products that cannot be further extended, removing the unincorporated selected modified nucleotide, extending those extension products that can be further extended, distinguishing those extension products that are further extended from those extension products that cannot be further extended, thereby detecting nucleotide variation in the first nucleic acid.
The invention further provides a method of detecting nucleotide variation within a first nucleic acid, comprising generating a set of single-stranded extension products in the presence of modified nucleotide bases and chain-terminating nucleotide bases from a first nucleic acid, wherein the extension products incorporate modified nucleotides that limit exonuclease activity to the 3xe2x80x2-terminal nucleotide base, and wherein the extension products have variable lengths, hybridizing the variable length extension products to a reference nucleic acid, contacting the hybridizing nucleic acids with an enzyme which can remove and replace the 3xe2x80x2-terminal nucleotide of the extension products in the presence of deoxynucleotide triphosphates, wherein the penultimate 3xe2x80x2-nucleotide is resistant to removal from the extension products, whereby extension products containing a penultimate 3xe2x80x2-nucleotide that does not hybridize with the corresponding position on the reference nucleic acid is not replaced with a nucleotide that hybridizes with the corresponding nucleotide on the reference nucleic acid and thereby cannot be further extended, extending those extension products that can be further extended, distinguishing those extension products that are further extended from those extension products that cannot be further extended, thereby detecting nucleotide variation in the first nucleic acid.